This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).

Animal experiments have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterin&#228;rdienst Bern (BE85/15).
1. Preparation of Xenopus Oocytes
<ol>
	<li>Maintain frogs (Xenopus laevis) on a 12 h/12 h light/dark cycle in water that is strictly kept at 20 &#176;C.</li>
	<li>Remove the lobes of the ovaries from female frogs9. 
	NOTE: The removal of the lobes is a stimulus for regeneration, and often the quality of the oocytes improves with surgery. The oocyte is covered by a vitelline layer, a layer of follicle cells, and connective tissue containing the blood vessels21. The entire structure is termed the &#34;follicle.&#34;</li>
	<li>Place the lobes of the ovaries that are densely packed with the follicles containing the oocytes in sterile&#160;Barth&#39;s Saline7 (MBS) supplemented with penicillin and streptomycin (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES (pH&#160;7.5, NaOH), 100 &#181;g/mL penicillin, and 100 &#181;g/mL streptomycin).</li>
	<li>Single-out stage V-VI21 follicles. Hold the ovary lobe with forceps. Lower the platinum loop (Figure 1) over the follicles and gently withdraw the loop to disrupt the connective tissue containing the follicle from the ovary tissue. 
	NOTE: Stage V-VI21 follicles are characterized by their size (1.0 - 1.2 mm in diameter) and by the good contrast between the dark pigmented animal hemisphere and the yellowish vegetal hemisphere.</li>
	<li>Transfer only healthy-looking follicles (i.e.&#160;perfectly spherical and with intact pigmentation) to a 35 mm diameter Petri dish by means of a plastic Pasteur pipette (cut the back to an opening diameter of 1.5 mm).</li>
</ol>
2. Microinjection of mRNA into the Cytoplasm
Note: The microinjection system described here is derived from that reported by Kressmann and Birnstiel22.
<ol>
	<li>Prepare cRNAs from the respective cDNAs coding for the subunits of the &#945;4&#946;2&#948; GABAA receptor by in vitro transcription, the addition of a poly (A+) tail, and RNA quantification by gel electrophoresis23.</li>
	<li>Prepare microinjection pipettes from borosilicate glass capillaries (1.0 mm outer diameter (OD), 0.58 mm inner diameter (ID), 100 mm length) using a micropipette puller. Break off the tips of the glass capillaries under a microscope using a micromanipulator and microfilament to create a tip diameter of 12 - 15 &#181;m with a beveled tip.</li>
	<li>Backfill the microinjection pipettes with paraffin oil using a 10 mL syringe, and then mount the microinjection pipette onto a homebuilt microinjection apparatus (Figure 2). 
	NOTE: The homebuilt oil hydraulic injection apparatus consists of a grill motor controlled by a foot pedal switch. An additional switch changes the turning mode of the motor. This motor drives a micrometer screw (0.5 mm/turn) that advances or retracts the plunger of a 10 &#181;L glass syringe. The injection is at a speed of about 3 rpm and a 0.5 turn corresponds to a 50 nL injection into a follicle. The tip of the syringe is connected to thick-walled polytetrafluoroethylene tubing that is itself connected to the injection needle by a very short piece of Tygon tubing. The injection capillary is held by a drill chuck that is controlled by a micromanipulator. The entire system is filled with paraffin oil. Any air bubbles should be avoided, as they will impair the injection system. The setup is lit with a cold light source. A stereomicroscope is required for optical control. Follicles are visualized under cold light with a stereomicroscope (40X magnification). Performance of the injection setup is tested by the injection of radioactive tracer into Xenopus oocytes. A volume of 45 - 55 nL should be delivered per injection.</li>
	<li>Using a pipette with a sterile plastic tip, test the setup by placing a droplet of sterile water onto the clean inside of a moisture-resistant thermoplastic (2 x 2 cm). Immerse the tip of the injection pipette into this droplet, and then turn the motor to retract the plunger (negative pressure inside the injection pipette). 
	NOTE: Water should enter the pipette and form a visible interface with the oil.</li>
	<li>Retract the tip of the pipette from the droplet and apply positive pressure to the inside of the injection pipette. A water droplet should form at the tip of the injection pipette.</li>
	<li>Prepare for the injection of the follicles by lining them up with the vegetal poles pointing upwards in the interstices of a nylon mesh (G: 0.8 mm) glued to the bottom of a Petri dish (60 mm) covered with MBS.</li>
	<li>Dispense 2 &#181;L of mRNA using a pipette with a sterile plastic tip onto the clean inside of a thermoplastic. Take the mRNA up into the injection pipette by applying negative pressure to the inside of the injection pipette.</li>
	<li>Position the injection pipette over an individual follicle using the micromanipulator.</li>
	<li>Insert the injection needle into the center of the vegetal pole and inject 50 nL of mRNA at a flow rate of 0.6 &#181;L/min by applying positive pressure to the inside of the pipette. Wait 5 - 10 s before removing the injection pipette tip from the follicle to avoid mRNA escape. 
	NOTE: The mRNA should be injected into the vegetal (yellowish) pole to avoid injection into the nucleus, which is located in the animal hemisphere.</li>
	<li>Transfer the injected follicles to a new Petri dish (35 mm) filled with 2 mL of MBS. Place the dish into a wine cooler set at 18 &#176;C. Incubate the injected follicles for 1 - 7 d before recording, depending on the identity of the newly expressed protein.</li>
</ol>
3. Stripping of the Follicles (Figure 3)
NOTE: As mentioned above, the oocyte covered by a vitelline layer, follicle cells, and connective tissue, which contains the blood vessels24, is known as a &#34;follicle.&#34; All layers except for the vitelline layer, which provides mechanical stability without preventing the access of solutions to the cell surface, must be removed before electrophysiological experiments. The follicle without the surrounding cell layers has previously been termed a &#34;denuded&#34; oocyte25. This step is usually performed on the same day as the electrophysiological experiments.
<ol>
	<li>Transfer ten injected follicles to a borosilicate glass tube containing 0.5 mL of MBS, 1 mg/mL collagenase, and 0.1 mg/mL trypsin inhibitor. Immerse the tube in a water bath maintained at 36 &#176;C. Incubate the follicles for 20 min and shake the tubes occasionally.</li>
	<li>Rinse the follicles by transferring them to a second tube containing 1 mL of MBS at RT. Leave them in the tube for about 10 s.</li>
	<li>Transfer the follicles to a third tube containing 0.5 mL of doubly concentrated MBS containing 4 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), and incubate them for 4 min at RT. Shake the tube occasionally. 
	NOTE: The hypertonic solution (doubly concentrated MBS) is used to induce shrinkage of the oocytes within follicle cell layers, thereby detaching follicular cell layers from the oocyte; EGTA is added to inhibit collagenase activity.</li>
	<li>Rinse the follicles by transferring them to a second tube containing 1 mL of MBS at RT. Leave them in the tube for about 10 s.</li>
	<li>Transfer the oocytes to a Petri dish (35 mm diameter) containing 2 mL of MBS. Under a stereomicroscope, separate the outer envelopes from the follicles by simply pushing the naked oocyte away using the platinum loop. 
	NOTE: The outer envelopes of the oocytes stick firmly to the culture dish and can be easily separated from the oocytes. Some oocytes will spontaneously lose their outer envelopes and will be recovered as denuded oocytes. This procedure results in a relatively stable preparation that keeps the spherical appearance of the oocytes.</li>
</ol>
4. Fast Solution Change around the Oocyte
<ol>
	<li>Perform a voltage clamp experiment, as described9,26.</li>
	<li>Change the solution of the gravity-fed perfusion system by turning the perfusion switches as required by the experiment.
	<ol>
		<li>Apply the perfusion solution at 6 mL/min through a glass capillary with an inner diameter of 1.35 mm, the mouth of which is placed about 0.4 mm from the surface of the oocyte, to allow fast changes in agonist concentration around the oocyte. 
		NOTE: The rate of change has been previously estimated as 70% in less than 0.5 s27.</li>
	</ol>
	</li>
</ol>

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<li>Gurdon, J. B. American clawed frogs. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGurdon%2C+JB+%22Methods+in+developmental+biology%22">Methods in developmental biology</a>. Wilt, F. H., Wessels, N. K. , Thomas Y. Crowell Co. New York. 75-84 (1967).</li>
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</ol>

The authors have nothing to disclose.

The methods described in this article deviate from those used traditionally7-9. It is standard to expose the lobes of the ovary to a 1 to 2 h collagenase treatment8; isolate undamaged, denuded oocytes; and inject them with mRNA using commercial injection devices. This classical procedure has the following drawbacks: 1) Oocytes are likely to be damaged by the long exposure to high concentrations of collagenase. 2) The unstable denuded oocytes must be stored until the experiment. 3) Denuded oocytes are more likely to suffer during microinjection than follicles. Commercial injection devices use large-diameter (about 20 &#181;m) injection needles, likely to result in a relatively high rate of damage. The advantages of the improved procedure described here are as follows: exposure to collagenase is limited to 20 min, denuded oocytes do not have to be stored, and the small tip diameter of the pipettes (about 12 - 15 &#181;m) used for microinjection does not require prior &#34;defolliculation&#34; of the follicles for the mRNA injection. The single critical step is that the follicle incubation temperature in collagenase solution should be carefully adjusted to 36 &#176;C and should not exceed it.
For solution changes in electrophysiological experiments, very often the solution in the measurement chamber is changed, which takes a substantial amount of time, depending upon size of the chamber and the perfusion rate. Using the perfusion capillary avoided this.
The major drawback of the expression of ion channels in Xenopus oocytes is their giant size. Voltage control faster than about 2 ms is difficult, and very fast (&#60;0.5 s) solution changes require elaborate procedures. Furthermore, strongly hydrophobic substances may almost be irreversibly bound to the egg yolk of the oocyte.
The methods outlined here have allowed us to investigate in a time-saving manner the functional properties of recombinant GABAA receptors, their modulation by synthetic compounds and plant substances, their subunit arrangement, and the location of drug binding sites in receptors of different subunit composition. The methods described here are suited for the expression of any protein of choice in the Xenopus oocyte. The very simple method for fast solution changes of the medium around the oocyte may be applied to the study of any ligand-gated ion channel or carrier.

Xenopus oocytes are widely used as an expression system (References 2 - 3, and references therein). They are able to properly assemble and incorporate functionally active multisubunit proteins into their plasma membranes. Using this system, it is possible to functionally investigate membrane proteins alone or in combination with other proteins, in order to study the properties of mutated, chimeric, or concatenated proteins, and to screen potential drugs.
Advantages of using oocytes over other heterologous expression systems include the simple handling of the giant cells, the high proportion of cells expressing foreign genetic information, the simple control of the environment of the oocyte by means of bath perfusion, and the control of the membrane potential.
The drawback of this expression system is the seasonal variation observed in many laboratories17-20. The reason for this variation is far from clear. Additionally, the quality of oocytes is often observed to vary strongly. Traditional methods7-9 have included the isolation of ovary lobes, the exposure of ovary lobes to collagenase for some h, the selection of denuded oocytes, and the oocyte microinjection. Here, a number of alternative, fast procedures are reported that have allowed us to work with this expression system for more than 30 years with no seasonal variation and little variation in oocyte quality.
The modified and improved methods described here for the isolation of oocytes, microinjection with cRNA, and removal of follicular cell layers can be used for the expression of any protein of choice in the Xenopus oocyte. The very simple method for fast solution changes of the medium around the oocyte may be applied to the study of any ligand-gated ion channel and of carriers.

<table><tbody><tr><td>NaCl</td><td>Sigma</td><td>71380</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P-9541</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>M-1880</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>223560</td><td></td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>C1396</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>Penicilin/streptomycin</td><td>Gibco</td><td>15140-148</td><td>100 &amp;mu;g penicillin/mL and 100 &amp;mu;g streptomycin/mL</td></tr><tr><td>Platinum wire loop</td><td>home-made</td><td></td><td></td></tr><tr><td>Micropipette puller</td><td>Zeitz-Instruments GmBH</td><td>DMZ</td><td></td></tr><tr><td>Hamilton syringe&amp;nbsp;</td><td>Hamilton</td><td>80300</td><td>10 &amp;mu;L, Type 701N</td></tr><tr><td>Thick walled polytetrafluoroethylene tubing</td><td>Labmarket GmBH</td><td></td><td>1.0 mm OD&amp;nbsp;</td></tr><tr><td>Paraffin oil</td><td>Sigma</td><td>18512</td><td></td></tr><tr><td>Nylon net, G: 0.8 mm&amp;nbsp;</td><td>ZBF Z&amp;uuml;richer Beuteltuchfabrik AG</td><td></td><td></td></tr><tr><td>Borosilicate glass tube&amp;nbsp;</td><td>Corning</td><td>99445-12</td><td>PYREX</td></tr><tr><td>Collagenase NB Standard Grade</td><td>SERVA</td><td>17454</td><td></td></tr><tr><td>Trypsin inhibitor type I-S</td><td>Sigma</td><td>T-9003</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3389</td><td></td></tr><tr><td>Glass capillary</td><td>Jencons (Scientific ) LTD.</td><td>H15/10</td><td>1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited</td></tr><tr><td>Borosilicate glass capillary</td><td>Harvard Apparatus Limited</td><td>30-0019</td><td>1.0 OD x&amp;nbsp; 0.58 ID x 100 Length mm (for microinjection)&amp;nbsp;</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>Harvard Apparatus Limited</td><td>30-0044</td><td>1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)</td></tr><tr><td>&amp;gamma;-Aminobutyric acid (GABA)</td><td>Sigma</td><td>A2129</td><td></td></tr><tr><td>3&amp;alpha;,21-Dihydroxy-5&amp;alpha;-pregnan-20-one (THDOC)</td><td>Sigma</td><td>P2016</td><td></td></tr><tr><td>grill motor</td><td>Faulhaber&amp;nbsp;</td><td></td><td>DC micromotor Type 2230 with gear Type 22/2</td></tr><tr><td>micrometer screw</td><td>Kiener-Wittlin</td><td>10400</td><td>TESA, AR 02.11201</td></tr><tr><td>Sterile plastic transfer pipettes</td><td>Saint-Amand&amp;nbsp; Mfg.</td><td>222-20S</td><td></td></tr></tbody></table>

xenopus oocytes: optimized methods for microinjection, removal of follicular cell layers, and fast solution changes in electrophysiological experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus oocytes were mechanically singled out using a platinum loop (Figure 1). The oocytes were microinjected with mRNA coding for the GABAA receptor subunits &#945;4, &#946;2, &#948;, 0.5:0.5:2.5 fmol/oocyte (Figure 2). After 4 d, follicular cell layers were removed (Figure 3). Oocytes were voltage clamped at -80 mV and exposed to increasing concentrations of &#947;-aminobutyric acid (GABA) in the presence of 1 &#181;M 3&#945;,21-dihydroxy-5&#945;-pregnan-20-one (THDOC), a potent positive allosteric modulator of the GABAA receptor. Figure 4A shows original current traces recorded in such an experiment. Figure 4B shows elicited current amplitude depending on the GABA concentrations. This allows determination of the sensitivity of this subunit combination toward GABA. The individual curves were fitted and standardized to Imax and subsequently averaged. The equation used was I(c)=Imax/(1+(EC50/c)n), where c is the concentration of GABA, EC50 the concentration of GABA (in the presence of 1 &#181;M THDOC) eliciting a half-maximal current amplitude, Imax is the maximal current amplitude, I is the current amplitude, and n is the Hill coefficient. The EC50 of the &#945;4&#946;2&#948; GABAA receptor amounted to 0.41 &#177; 0.12 &#181;M, and n was 0.76 &#177; 0.04.
<img alt="Figure 1" src="/files/ftp_upload/55034/55034fig1.jpg" /> 
Figure 1: Platinum Loop. The platinum wire loop mentioned in protocol step 1.4. Platinum is a metal that can be bent without producing splinters, and it does not stick to biological material. <a href="http://cloudflare.jove.com/files/ftp_upload/55034/55034fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55034/55034fig2.jpg" /> 
Figure 2: Homebuilt Microinjection Apparatus. The apparatus is described in protocol step 2. a, grill motor; b, micrometer screw; c, spring between syringe and plunger; d, 10 &#181;L glass syringe; e, thick-walled polytetrafluoroethylene tubing; f, hand drill chuck; g, injection pipette; h, stereomicroscope; and i, motor control. <a href="http://cloudflare.jove.com/files/ftp_upload/55034/55034fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55034/55034fig3.jpg" /> 
Figure 3. Scheme Showing the Defolliculation of an Oocyte. Oocytes are first incubated in a collagenase solution in a water bath at 36 &#176;C for 20 min. Oocytes are then washed in a modified Barth medium. The final incubation step is in a hypertonic EGTA solution at RT&#160;for 4 min. As a result, the connective tissue and the follicle cells are removed to give way to the denuded oocyte. <a href="http://cloudflare.jove.com/files/ftp_upload/55034/55034fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55034/55034fig4.jpg" /> 
Figure 4. Current Traces from a Two-electrode Voltage Clamp Experiment. A, Current traces from a GABA concentration response curve in the presence of 1 &#181;M THDOC obtained from a Xenopus oocyte expressing the &#945;4&#946;2&#948; GABAA receptor. The bars indicate the time period of GABA/1 &#181;M THDOC perfusion. Increasing concentrations of GABA were applied to the oocytes, and the corresponding current amplitudes were determined. GABA concentrations are indicated above the bars. B, Averaged concentration response curves of the &#945;4&#946;2&#948; GABAA receptor. Individual curves were first normalized to the fitted maximal current amplitude and were subsequently averaged. The data are shown as mean &#177; SD, n = 3. <a href="http://cloudflare.jove.com/files/ftp_upload/55034/55034fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments at JoV

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

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20.1K Views.  University of Bern. Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented. 

Video: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Developmental Biology

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Biochemistry

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Summary

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Functional Cloning Using a Xenopus Oocyte Expression System

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

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Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

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